Regulatory properties of citrate synthase from Rickettsia prowazekii.
Citrate synthase [citrate (si)-synthase] (EC 4.1.3.7) was partially purified from extracts of highly purified typhus rickettsiae (Rickettsia prowazekii). Molecular exclusion and affinity column chromatography were used to prepare 200-fold-purified citrate synthase that contained no detectable malate dehydrogenase (EC 1.1.1.37) activity. Rickettsial malate dehydrogenase also was partially purified (200-fold) via this purification procedure. Catalytically active citrate synthase exhibited a relative molecular weight of approximately 62,000 after elution from a calibrated Sephacryl S-200 column. Acetyl coenzyme A saturation of partially purified enzyme was sensitive to strong competitive inhibition with adenylates (ATP greater than ADP much greater than AMP). [beta,gamma-methylene]ATP, dATP, and dADP also caused strong inhibition, but guanosine and cytosine nucleotides were significantly less inhibitory. Adenylates had no effect on oxalacetate saturation kinetics when acetyl coenzyme A was present in high concentration (greater than or equal to 50 microM). Neither NADH nor alpha-ketoglutarate affected the saturation kinetics of rickettsial citrate synthase. Thus, citrate synthase from R. prowazekii exhibits greater similarity to the eucaryotic and gram-positive procaryotic enzymes than to citrate synthase from free-living gram-negative bacteria. These results represent the first characterization of a highly purified key regulatory enzyme from these obligate intracellular parasitic bacteria.
D000105:Acetyl Coenzyme A / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002950:Citrate (si)-Synthase / Q000302:isolation & purification / Q000378:metabolism*; D007656:Ketoglutaric Acids / Q000494:pharmacology; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D007652:Oxo-Acid-Lyases / Q000378:metabolism*; D012283:Rickettsia prowazekii / Q000201:enzymology*
D007656:Ketoglutaric Acids; D009243:NAD; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000105:Acetyl Coenzyme A; D000255:Adenosine Triphosphate; D002950:Citrate (si)-Synthase; D007652:Oxo-Acid-Lyases